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The role of hepatocyte growth factor （HGF）-induced activation （phosphorylation） of its receptor, c-Met, and subsequent downstream signaling during muscle regeneration has not yet been adequately determined. We examined HGF and phospho-c-Met expression and localization during the expansion phase and time course of satellite cell activation, which is critical for muscle regeneration after injury. HGF, c-Met, myogenic differentiation 1 （MyoD）, and myogenin mRNA levels were significantly increased during muscle regeneration after injury. Phospho-c-Met was localized on the surface of regenerating muscle cells. HGF was colocalized in the same cells as phospho-c-Met, MyoD, or myogenin. The muscle area occupied by HGF-, phospho-c-Met-, MyoD-, or myogenin-positive cells expanded along with expansion of regenerating muscle. Thus, c-Met is expressed in regenerating muscle cells immediately after injury, and its kinase activity is likely activated by HGF expressed by the same cells, suggesting that HGF and c-Met contribute to myogenesis in vivo.

The research performed at the Department of Urology Osaka University Graduate School of Medicine is overviewed. Renal ischemia-reperfusion (I/R) injury is inevitable in transplantation and is related to longterm graft function. MF-1, a bifunctional hepatocyte growth factor-macrophage stimulating protein chimera, was found to prevent apoptosis. In our study, MF-1 directly guarded cultured proximal tubular epithelial cells from hypoxia-induced necrosis and apoptosis in vitro, and MF-1 treatment ameliorated renal dysfunction by preventing apoptosis in rat I/R injury model. The erythropoietin molecule modified by carbamylation (CEPO) has been identified and was demonstrated to protect several organs without increasing the hemoglobin concentration. The therapeutic effect of CEPO was evaluated using an endothelial tube formation assay, and a rat ischemia-reperfusion injury model. CEPO treatment induced more capillarylike formation than EPO. CEPO-treated kidneys showed minimal tubular apoptosis with increased peritubular capillary endothelial cells. We identified a new therapeutic approach using CEPO to protect the kidney from ischemia-reperfusion injury by promoting angiogenesis.

[抄録] 背景：長期にわたる腹膜透析では腹膜線維化により腹膜機能が低下し，これが原因で腹膜透析を中止することがある．腹膜線維化にはtransforming growth factor-beta 1 (TGF-β1)とhepatocyte growth factor (HGF)の不均衡が密接に関わっているとの報告がある．現在までにわれわれは，外因性HGFが，高濃度グルコースやTGF-β1による傷害を受けたヒト腹膜中皮細胞の増殖能を改善することを報告してきた．今回われわれは腹膜硬化症モデルラットにおけるHGFの効果を検討した．方法：ラットに0.1%グルコン酸クロルヘキシジンを35日間腹腔内投与し，腹膜硬化症モデルラットを作成した．ラット腹膜中皮細胞にヒトHGF遺伝子を導入して腹膜硬化症モデルラット腹腔内へ投与し，この時の組織学的変化を検討した．結果：腹膜硬化症モデルラットにおける腹膜の組織学的変化として，中皮細胞の脱落と線維芽細胞の増加，新生血管の増生を認めた．これらの変化はヒトHGF遺伝子導入ラット腹膜中皮細胞を腹腔内投与することで抑制された．結論：ヒトHGF遺伝子導入ラット腹膜中皮細胞の腹腔内投与により腹膜線維化が抑制された．, 本文データはCiNiiから複製したものである。

Human hepatocyte growth factor (HGF) was first discovered in plasma from fulminant hepatic failure as a potent mitogen for adult hepatocytes in 1985, and it was purified from the patients' plasma in 1986 in our laboratory. However, subsequent studies have revealed that HGF has various physiological activities such as mitogen, motogen, morphogen and angiogen to most of epithelial and endothelial cells. Thus, HGF is now known as a broad-spectrum and multifunctional cytokine involved in a variety of physiological processes, including tissue development, regeneration and wound healing. In this review, a history how HGF was discovered is described. It is also described the physiological roles of HGF, especially in oral biology.

The oral and craniofacial regions consist of a variety of tissues including highly differentiated cells originating in different germinal layers. During embryonic development, cell growth factors control migration, proliferation, and differentiation of these cells. Hepatocyte growth factor (HGF) is expressed in the first branchial arch, an anlage of oral tissues. The present review describes the multiple, important roles of HGF in the development of Meckel's cartilage, mandibular bone, teeth, and the tongue as shown by immunohistochemical techniques, RT-PCR, and antisense inhibition experiments using mandibular organ culture models. The results of these experimentssuggest that i) HGF promotes the proliferation of osteoblasts and their progenitor cells, and initiates increases in mandibular volume by membranous ossification, ii) HGF promotes the proliferation of chondrocytes of Meckel's cartilage and accelerates cartilaginous growth in length, iii) The degeneration of Meckel's cartilage with chondrocytes hypertrophy is induced by the decrease in endogenous HGF during the later stage of development, iv) HGF is essential for the migration of myoblasts in the tongue during the early stage of development, and aglossia is induced by inhibition of myoblast migration, v) HGF promotes myoblast proliferation in the tongue.

ヒト前立腺癌細胞株であるDU145は前立腺間質細胞からのHGF産生を促進する何らかのHGF-inducerを分泌し, その増加した間質細胞由来のHGFがDU145の浸潤能を促進した.このような前立腺癌細胞の独特な癌-間質相互作用において, HGFが癌細胞の浸潤を制御する主要因子の一つである可能性が示唆された, We examined how prostate stromal cell-derived hepatocyte growth factor (HGF) affects invasion of prostate cancer cells through tumor-stromal interaction. The effects of HGF, various growth factors [transforming growth factor (TGF)-alpha, TGF-beta 1, basic fibroblast growth factor, keratinocyte growth factor, and platelet-derived growth factor], and conditioned medium (CM) from prostate stromal cells (PrSC) on prostate cancer cells (LNCaP, PC-3 and DU145) were determined by collagen gel invesion assay. DU145 cells and PrSC were co-cultured for matrigel invasion chamber assay. LNCaP and PC-3 cells did not respond to any of the factors examined. Invasion of DU145 cells into the collagen gel matrix was induced by HGF and TGF-beta 1, but not by any of the other factors tested. When DU145 cells were cultured in CM from PrSC or co-cultured with PrSC, the cells acquired invasive potential, and this invasion was inhibited by an antibody against HGF, but not against TGF-beta 1. Induction activity of CM from cancer cells to stimulate HGF production by PrSC was studied by ELISA method and Western blotting. Native type HGF production in PrSC was enhanced by some unknown inducer(s) produced by cancer cells. In summary, PrSC-derived HGF enhanced invasive activity of the prostate cancer cell line DU145 through tumor-stromal interaction wherein DU145 cells secreted some HGF-inducer(s) for PrSC.

To observe the effect of Hepatocyte growth factor/scatter factor (HGF/SF) on the invasion and metastasis of human oral squamous cell carcinoma (SCO cells, the invasive ability and internal signal transduction were examined The invasive ability of tumor cells (SAS, HSC-3,Ca9-22) was enhanced dose-dependently by HGF/SF stimulation, and C3 exoenzyme which is a specific inhibitor of Rho activation significantly reduced the invasive ability of SAS cells The HGF/SF stimulation enhanced the tyrosin phosphorylation of HGF receptors (c-Met) and focal adhesion kmase (FAK^) of SAS cells, and C3 exoenzyme completely inhibited the activation of FAK^ It was observed that Rho protein located around the nuclear area translocated to the membrane area and subsequently increased in the cytolysate, following HGF/SF stimulation These results suggest that the activation of FAK^ caused by phosphorylation of c-Met may enhance the HGF/SF, inducing invasive ability and that Rho protein possibly regulates the activation of FAK^ through the translocation itself

We found the existence of a hepatotrophic factor in plasma or sera of patients with fulminant hepatic failure, and have recently purified a hepatocyte growth-promoting factor, human hepatocyte growth factor (hHGF) from the plasma obtained during the plasma exchange therapy of the patients. The hHGF level in plasma changed parallel with the coma grade of the patients, and it rapidly decreased to almost the normal level when the patient recovered from the disease. Thus, the plasma hHGF level seems to be the result of a self-defense mechanism and hHGF may be a physiological hepatotrophic factor for human liver regeneration. In this review, we summarized our recent papers on hHGF including purification and characterization of hHGF, amino acid sequence of hHGF deduced from its cDNA sequence and plasma levels of hHGF in patients with liver diseases determined by an enzyme-linked immunosorbent assay using polyclonal and monoclonal antibodies against hHGF. Because the amino acid sequence of hHGF has high homology to some serum proteins involved in blood coagulation and fibrinolysis such as plasminogen, we also discussed and predicted the secondary structure of hHGF from that of human plasminogen.

Background: Expression of neurotrophins (NTs) and their receptors is increased during hepatic regeneration, but their role is not well understood. Methods: NTs and their receptors were investigated by RT-PCR and immunostaining in regenerating livers after two-thirds hepatectomy (PH) and in hepatocytes and hepatic stellate cells (HSCs) isolated from regenerating livers in mice. Induction of apoptosis after treatment with NGF and the expression of α-smooth muscle actin (SMA), interleukin 6 (IL-6) and hepatocyte growth factor (HGF) were also investigated in regenerating HSCs. Results: Nerve growth factor (NGF) and p75 NT receptor (p75NTR) mRNA were elevated after PH, while other NTs and NT receptors showed no remarkable change. NGF was detected in regenerating hepatocytes, but not in normal hepatocytes. Regenerating HSCs expressed increased p75NTR and SMA in vivo and showed an activated phenotype and the high expression of HGF and IL-6 in vitro. Enhanced cell death was seen in HSCs, both from normal and regenerating liver, after treatment with NGF. Conclusions: Although activated HSCs may produce the factors that regulate liver regeneration, the de novo NGF production by regenerating hepatocytes may induce the death of activated HSCs via p75NTR, leading to termination of hepatic regeneration.

研究種目:科学研究費補助金基盤研究(C), 報告年度:2000年度, 研究課題番号:11670567, 研究概要:ヒト肺小細胞癌株(Wa)とその間質線維芽細胞(W-fib)の相互作用について検討した。W-fibをfeederとして共培養した場合のplating efficiencyは、Waに比べ有意に亢進し、ヌードマウス皮下腫瘍形成でも、Wa単独接種に比べ、W-fibを同数混じて接種する場合では、腫瘍形成潜時の有意な短縮を認めた。以上より、in vitro、in vivoにおいて、Waの腫瘍形成に対し、その間質細胞であるW-fibは促進作用があると思われる。次に、W-fibに、in vitroでβ-gal遺伝子を導入し、W-fibを特異的に染色同定できるように処理した後、WaとW-fibを混じでヌードマウス皮下に接種し、腫瘍形成過程における線維芽細胞の役割を形態的に観察することに成功した。腫瘍が島状に増殖し、β-gal陽性の線維芽細胞はそれを取り巻くように分布し、また腫瘍内の血管に沿ってもその分布が確認された。WaとW-fibの相互作用のメカニズムを解析する目的で、培養上清中の各種増殖因子をELISAにより測定した。共培養開始18時間後に、培養上清中のHGFは一旦著増し、48時間以内に消失した。HGFのピークに引き続き、VEGFが数日にわたって著増した。一方、Wa単独、W-fib単独培養では、HGF、VEGFとも検出されなかった。さらにHFGはW-fibから、VEGFは主にWaから産生されることが判明した。HGFに対するアンチセンスオリゴDNAを培養上清に添加することにより、VEGF産生、Waのplating efficiency の亢進効果がブロックされることが確認された。…

研究種目:科学研究費補助金基盤研究(C), 報告年度:2000年度, 研究課題番号:11670567, 研究概要:ヒト肺小細胞癌株(Wa)とその間質線維芽細胞(W-fib)の相互作用について検討した。W-fibをfeederとして共培養した場合のplating efficiencyは、Waに比べ有意に亢進し、ヌードマウス皮下腫瘍形成でも、Wa単独接種に比べ、W-fibを同数混じて接種する場合では、腫瘍形成潜時の有意な短縮を認めた。以上より、in vitro、in vivoにおいて、Waの腫瘍形成に対し、その間質細胞であるW-fibは促進作用があると思われる。次に、W-fibに、in vitroでβ-gal遺伝子を導入し、W-fibを特異的に染色同定できるように処理した後、WaとW-fibを混じでヌードマウス皮下に接種し、腫瘍形成過程における線維芽細胞の役割を形態的に観察することに成功した。腫瘍が島状に増殖し、β-gal陽性の線維芽細胞はそれを取り巻くように分布し、また腫瘍内の血管に沿ってもその分布が確認された。WaとW-fibの相互作用のメカニズムを解析する目的で、培養上清中の各種増殖因子をELISAにより測定した。共培養開始18時間後に、培養上清中のHGFは一旦著増し、48時間以内に消失した。HGFのピークに引き続き、VEGFが数日にわたって著増した。一方、Wa単独、W-fib単独培養では、HGF、VEGFとも検出されなかった。さらにHFGはW-fibから、VEGFは主にWaから産生されることが判明した。HGFに対するアンチセンスオリゴDNAを培養上清に添加することにより、VEGF産生、Waのplating efficiency の亢進効果がブロックされることが確認された。…